A NOVEL CALCIUM-BINDING PROTEIN IN AMNIOTIC-FLUID, CAAF1 - ITS MOLECULAR-CLONING AND TISSUE DISTRIBUTION

We found by using a Ca-45(2+) overlay technique a large amount of Ca2-binding activity in bovine amniotic fluid from which a novel calcium- binding protein (CaBP) was purified and is referred to as CAAF1 (calci um-binding protein in amniotic fluid-1), with an apparent molecular ma ss of 8 kDa determined by N-tris(hydroxymethyl)-methylglycine/SDS-PAGE . It was structurally homologous with MRP/calgranulin proteins (MRP8/c algranulin A and MRP14/calgranulin B), members of the S100 protein fam ily, which are abundantly found in the cytoplasm of granulocytes and m acrophages. CAAF1 lacked the predicted signal peptide sequence, which is consistent with other CaBPs. The tissue and cellular distribution o f CAAF1 was determined by monoclonal antibodies developed against this protein. Its immunoreactivity was found in squamous epithelial cells, neutrophils, and some macrophages throughout the fetal body. An espec ially characteristic staining pattern was obtained in the squamous epi thelium, including that of the esophagus, skin and amnion: CAAF1 was d etected in the suprabasal squamous epithelial cells undergoing differe ntiation, but not in the cells in the proliferating basal layer. North ern blot analysis also showed that CAAF1 mRNA was highly expressed in bovine fetal esophagus and skin. On the other hand, our ELISA studies showed that CAAF1 protein was present in amniotic fluid at a concentra tion of about 120 nM, which was over 30 times as high as that in the f etal serum. These results suggested that CAAF1 is one of the stage-spe cific proteins in the differentiation of squamous epithelial cells, an d that CAAF1 is preferentially produced by fetal squamous epithelial c ells, including epidermal keratinocytes and amniotic epithelial cells, and it is stored in the amniotic fluid during embryogenesis.